低氧诱导的外泌体OTUB1调控血管瘤内皮细胞生物学行为的作用及机制研究

Hypoxia is a common microenvironment in the proliferating infantile hemangiomas (IHs). However, the critical role of hypoxia in the development of IHs is still unclear. Recent studies have shown that exosomes secreted by hypoxic tumor stem cells could deliver self-biological information into surrounding tumor cells, which could favor in the tumor progression. Our preliminary study indicated that exosomes stimulated by hypoxia participated in the intracellular communications between hemangioma stem cells (Hemscs) and hemangioma endothelial cells (Hemecs), and exosomes derived from hypoxic Hemscs could promote the proliferation and angiogenesis of Hemecs in vitro. Moreover, the mRNA levels of ovarian tumor domaincontaining ubiquitin aldehyde-binding protein 1 (OTUB1) were obviously elevated in hypoxic Hemsc-exosomes, which were closely associated with the up-regulation of estrogen receptor alpha (ERɑ) proteins. On basis of these findings, we hypothesized that hypoxia could induce the expression of OTUB1 in Hemscs and deliver OTUB1 mRNA to surrounding Hemecs by exosomes. Exosomal OTUB1 mRNA derived from Hemscs could facilitate in the up-regulation of ERɑ, which would favor in the rapid proliferation and angiogenesis of Hemecs through ERɑ-induced pathway. To identify this hypothesis, we would employ human sample, cell and mice model to explore the molecular mechanisms underlying the biological behaviors of Hemecs regulated by exosomal-OTUB1 induced by hypoxia, and further uncover the role of hypoxia regulating the cellular microenvironment in IHs. Our project would contribute to understand the unique biological features of IHs and provide a novel inspiration for treating vascular tumors.

低氧是婴幼儿血管瘤组织中的常见现象，但其调控血管瘤发生、发展的机制仍不明确。研究表明，低氧状态下肿瘤干细胞可通过外泌体途径将自身生物学信息传递给周围肿瘤细胞，促进肿瘤的发展。我们研究发现：低氧诱导的血管瘤干细胞外泌体可明显提高内皮细胞的增殖及血管生成能力；低氧血管瘤干细胞外泌体中去泛素化酶OTUB1 mRNA表达明显升高，且OTUB1的表达与内皮细胞中ERɑ上调有关。因此，我们提出假设：低氧微环境可诱导血管瘤干细胞中OTUB1表达增加，并通过外泌体将OTUB1 mRNA转运至周围内皮细胞，上调ERɑ信号通路，促使内皮细胞快速增殖，血管生成增加，从而促进血管瘤的发展。本课题拟从临床标本、体外细胞、动物模型三个层面探索低氧诱导的外泌体OTUB1调控血管瘤内皮细胞生物学行为的作用机制，进一步阐明低氧外泌体在血管瘤发展中的作用。本课题有助于加深对血管瘤发病机制的认识，并为血管瘤的治疗提供新的思路。

婴幼儿血管瘤是婴幼儿中最常见的血管源性肿瘤，但其发生发展机制依旧不明，临床治疗效果不佳。为寻找新的有效的治疗靶点，改善治疗效果，本课题从外泌体调控泛素/去泛素化的角度研究婴幼儿血管瘤的发生发展机制。从临床病例中获取血管瘤组织并分离培养血管瘤干细胞及血管瘤内皮细胞。提取血管瘤干细胞来源外泌体，鉴定后进行转录组测序，筛选出其中可能在血管瘤发生发展中发挥重要作用的去泛素化酶OTUB1。通过体内及体外试验，证实OTUB1具有促进血管瘤发生发展的功能。对血管瘤内皮细胞进行转录组测序及蛋白组学分析，预测TGFBI为OTUB1发挥促瘤作用的下游底物蛋白。进一步验证TGFBI与OTUB1之间的蛋白相互作用，结果表明，OTUB1可与TGFBI直接结合，去泛素化调控TGFBI，降低其泛素化水平，提高其蛋白稳定性。质谱分析及构建K22，25R突变型TGFBI证实TGFBI的泛素化位点位于K22和K25。为探明OTUB1对TGFBI的调控机制，针对OTUB1的酶依赖活性和非酶依赖途径调控方式，构建OTUB1的C91A与D88A突变体，证实OTUB1对TGFBI的去泛素化调控不依赖于OTUB1的去泛素化酶活性。在体内和体外回复实验中发现，TGFBI是OTUB1发挥促瘤作用的重要下游蛋白，且这种调控作用是通过影响血管瘤内皮细胞的糖酵解水平实现的。OTUB1可通过非酶活性依赖途径去泛素化TGFBI调控血管瘤内的糖酵解水平，促进血管瘤的发生发展，是十分具有潜力的治疗靶点，具有广阔的临床转化前景。